The present invention relates generally to immunoassays for lipoprotein(a) and more particularly to improved assays for the detection and quantification of lipoprotein(a) in fluid samples.
Lipoprotein(a) (Lp(a)) is found in human plasma in concentrations ranging from less than 1 to more than 100 mg/dL. Lp(a) closely resembles low density lipoprotein (LDL); both have a similar lipid composition and both include an apolipoprotein B-100 (apo B) component. The protein component of Lp(a), however, includes, in addition to one molecule of apo B, two molecules of a second protein designated apolipoprotein(a) (apo(a)) per Lp(a) particle. These two proteins are covalently linked by disulfide bond(s) that can be readily reduced. The function of Lp(a) is unknown; however, a significant correlation has been established between elevated Lp(a) levels, coronary artery disease, and the progression of atherosclerotic lesions.
A number of assay procedures for quantitating Lp(a) in fresh and frozen serum have been developed, as summarized by Morrisett, et al., at Ch. 4, pp. 129-152 in Plasma Lipoproteins, (A. M. Gotto, Jr., Ed.) Elsevier Science B.V. (Biomed. Division) (1987). These assays range in sensitivity and, in general, are based upon an immunochemical reaction between the apo(a) antigen and antibodies directed against one or more apo(a) determinants. Gaubatz, et al., J. Biol. Chem., 258:4582 (1983) describe an electroimmunoassay having a sensitivity range of 1-10 mg/dL. Albers, et al., J. Lipid Res., 18:331-338 (1977) describe a radioimmunoassay sensitive to 0.5 mg/dL. Albers and Hazzard, Lipids, 9:15-26 (1974) also quantitated Lp(a) by radial immunodiffusion. The lower limit of sensitivity of this assay was 1.5 mg/dl. Vu-Dac, et al., J. Lipid Res., 26:267-269 (1985) describe a latex particle immunoassay able to detect serum Lp(a) ranges of from 0.005 to 0.115 mg/dL. Duvic, C. R., et al., J. Lipid Res., 26:540-548 (1985), describe a highly sensitive enzyme-linked assay (ELISA) reported to be sensitive over the range of 0.001-0.140 mg/dL. According to this procedure, excess Lp(a) is absorbed to the well plate surface and mouse monoclonal antibody is competitively bound between the Lp(a) absorbed to the plate and that present in a test sample. Alkaline phosphatase-conjugated anti-mouse IgG is added and paranitro phenyl phosphate is provided as the phosphatase enzyme substrate. Enzymatically released para-nitro phenol is measured spectrophotometrically at 405 nm to provide for indirect quantification of the Lp(a) in the sample.
Also described in Morrisett, et al., supra, is a summary of the work of Gaubatz, J. W., et al. (assertedly submitted for publication) relating to a "sandwich" ELISA for Lp(a). Briefly, goat-anti-human apo(a) is bound to the surface of a plastic microtiter plate to which a Lp(a)-containing sample is added. A second antibody, rabbit anti-human apo(a) serum, is then added to form a sandwich with the Lp(a) immobilized by the first antibody, to wit: plate--goat antibody--Lp(a)--rabbit antibody. A peroxidase-conjugated anti-rabbit IgG antibody, is added and oxidization of an o-phenylenediamine substrate to a colored compound is measured spectrophotometrically at 492 nm. The increase in absorbance is proportional to the amount of Lp(a) present in the triple antibody complex and the assay is reported to have a sensitivity as low as 5.7 mg/dL.
Because all of the above-described assays are based on detection of the presence of apo(a) antigenic determinants in a serum sample, they also potentially "measure" serum proteins having amino acid sequence homology to apo(a). Recent efforts by the present inventors and their co-workers in determining a partial amino acid sequence for apo(a) have revealed that it is homologous to the serum protein plasminogen, a member of a protein superfamily composed of regulatory proteases of the fibrinolytic and blood coagulation systems. Eaton, et al., Proc. Nat'l. Acad. Sci. (USA), 84:3224-3228 (1987). Cross reactivity of Lp(a) and plasminogen with an anti-apo(a) antibody and with an anti-human plasminogen antibody demonstrates that apo(a) and plasminogen in fact share common epitopes, supporting the conclusion that the above-described Lp(a) assays based on detection of the apo(a) moiety of Lp(a) also "measure" any plasminogen present in a given sample. The Lp(a) concentration values obtained using the above described Lp(a) assays are therefore erroneously high. Thus, there exists a need in the art for a quantitative immunoassay for Lp(a) which is not sensitive to the presence of plasminogen in a serum sample.